Isolation of Human NK Cells and Generation of LAK Activity

Theresa L. Whiteside1

1 University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.7
DOI:  10.1002/0471142735.im0707s17
Online Posting Date:  May, 2001
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Abstract

This unit describes the preparation of natural killer (NK) cells from normal human peripheral blood and their incubation in vitro in the presence of interleukin 2 (IL‐2) to yield lymphokine‐activated killer (LAK) cells. A protocol is presented for isolating highly purified NK cell populations from PBMC, and another method presents steps for generating LAK cells from these purified NK cells via IL‐2 stimulation. An alternate protocol describes the generation of LAK cells directly from whole, unseparated PBMC preparations instead of purified NK populations. The caveat with the alternate protocol is that LAK activity generated in this manner represents the total cytotoxic potential of all LAK precursor cells‐‐i.e., all those PBMC that are capable of responding to IL‐2 by up‐regulation of cytotoxicity against NK‐resistant targets. In PBMC, LAK precursor cells are found among subpopulations of both NK cells and T lymphocytes.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Purified Human NK Cells from PBMC
  • Basic Protocol 2: Activation of NK Cells with IL‐2 to Generate LAK Activity
  • Alternate Protocol 1: Generation of LAK Activity from PBMC
  • Support Protocol 1: Titration of Monoclonal Antibodies for Isolating NK Cells by Negative Selection
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Purified Human NK Cells from PBMC

  Materials
  • Heparinized whole blood obtained via Vacutainer (Becton Dickinson) or a leukopak (cell pack from a leukapheresis donor, obtained from a blood bank)
  • recipePBS or recipeHBSS ( appendix 2A)
  • recipeRPMI‐2 medium (see recipe), 4°C and 37°C
  • recipeRPMI‐10 medium (see recipe), 37°C
  • MAbs specific for cells to be depleted, at optimal concentrations determined by titration (see protocol 4)
  • Magnetic beads coated with goat anti–mouse IgG (BioMag, PerSeptive Biosystems; store at 4°C)
  • recipeSterilized nylon wool columns (see recipe)
  • Luer‐Lok 3‐way disposable stopcocks
  • Luer‐Lok 19‐G needles
  • 50‐ml conical screw‐cap polypropylene centrifuge tubes
  • Sorvall RC‐3B or RT‐6000 centrifuge with H‐2000B or H‐1000B swinging‐bucket rotors (or equivalent)
  • 14‐ml round‐bottom screw‐cap polystyrene tubes
  • Strong magnet or magnetic cell sorter (MACS; e.g., Dynal, PerSeptive Biosystems, or Miltenyi Biotec)
  • Additional reagents and equipment for isolating PBMC by Ficoll‐Hypaque gradient centrifugation (unit 7.1), checking number of viable cells by trypan blue exclusion ( appendix 3B), and preparing sterilized nylon wool columns (unit 3.2)

Basic Protocol 2: Activation of NK Cells with IL‐2 to Generate LAK Activity

  Materials
  • Isolated NK cells (see protocol 1)
  • 6000 IU/ml recombinant human IL‐2 (rhIL‐2) in recipeRPMI‐10AB medium (see recipe), 37°C
  • recipeRPMI‐2AB (see recipe), room temperature
  • recipeRPMI‐10AB medium (see recipe), 37°C
  • recipeRPMI‐2 medium (see recipe)
  • 24‐well flat‐bottom tissue culture plates (Linbro, from ICN Biomedicals)
  • 15‐ml conical polypropylene tubes
  • Sorvall RC‐3B or RT‐6000 centrifuge with H‐2000B or H‐1000B swinging‐bucket rotors (or equivalent)
  • Additional reagents and equipment for determining number of viable cells by trypan blue exclusion ( appendix 3B)

Alternate Protocol 1: Generation of LAK Activity from PBMC

  • Heparinized whole blood obtained via Vacutainer (Becton Dickenson)
  • recipeHBSS ( appendix 2A)
  • recipePBS ( appendix 2A), room temperature and ice‐cold
  • recipeEDTA/PBS (see recipe), ice‐cold
  • recipeSerum‐free RPMI medium with L‐glutamine, HEPES, and antibiotic ( appendix 2A), ice‐cold
  • recipeRPMI‐10 medium (see recipe), ice‐cold
  • 15‐ml polypropylene conical tubes
  • Inverted microscope
  • Additional reagents and equipment for isolating PBMC by Ficoll‐Hypaque centrifugation (unit 7.1) and determining number of viable cells by trypan blue exclusion ( appendix 3B)
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Figures

Videos

Literature Cited

Literature Cited
   Caligiuri, M.A., Zmuidzinas, A., Manley, T., Levine, H., Smith, K.A., and Ritz, J. 1990. Functional consequences of interleukin 2 receptor expression on resting human lymphocytes: Identification of a novel natural killer cell subset. J. Exp. Med 171:1509‐1526.
   Grimm, E.A., Robb, R.J., Roth, J.A., Neckers, L.M., Lachman, L.B., Wilson, D.J., and Rosenberg, S.A. 1983. Lymphokine‐activated killer (LAK) cell phenomenon. III. Evidence that IL‐2 is sufficient for direct activation of peripheral blood lymphocytes into lymphokine‐activated killer cells. J. Exp. Med 158:1356‐1361.
   Julius, M.H., Simpson, E., and Herzenberg, L.A. 1973. A rapid method for the isolation of functional thymus‐derived murine lymphocytes. Eur. J. Immunol 3:645‐649.
   Nagler, A., Lanier, L.L., Cwirla, C., and Phillips, J.H. 1989. Comparative studies of human FcRIII‐positive and negative natural killer cells. J. Immunol 143:3183‐3191.
   Ortaldo, J.R., Mason, A., and Overton, R. 1986. Lymphokine‐activated killer cells: Analysis of progenitors and effectors. J. Exp. Med 164:1193‐1198.
   Phillips, J.H., Gemlo, B.T., Myers, W.W., Rayner, A.A., and Lanier, L.L. 1987. In vivo and in vitro activation of natural killer cells in advanced cancer patients undergoing continued recombinant interleukin 2 and LAK cell therapy. J. Clin. Oncol 5:1933‐1941.
   Pricop, L., Galatiuc, C., Manciuela, M., DeLeo, A., Sulica, A., Herberman, R.B., and Whiteside, T.L.. 1991. Expression of Fcµ receptors on human natural killer cells. Clin. Immunol. Immunopathol 59:355‐367.
   Rabinowich, H., Sedlmayr, P., Herberman, R.B., and Whiteside, T.L. 1993. Response of human NK cells to IL‐6. Alterations of the cell surface phenotype, adhesion to fibronectin and laminin, and tumor necrosis factor‐α/β secretion. J. Immunol 150:4844‐4855.
   Roberts, K., Lotze, M.T., and Rosenberg, S.A. 1987. Separation and functional studies of the human lymphokine‐activated killer cell. Cancer Res 47:4366‐4371.
   Siegel, J.P., Sharon, M., Smith, P.L., and Leonard, W.J. 1987. The IL2 receptor beta chain (p75): Role in mediating signals for LAK, NK and proliferative activities. Science 238:75‐78.
   Whiteside, T.L. and Herberman, R.B. 1990. Characteristics of natural killer cells and lymphokine‐activated killer cells. Their role in the biology and treatment of human cancer. Immunol. Allergy Clin. N. Amer 10:663‐704.
Key Reference
   Whiteside and Herberman, 1990. See above.
  Review of general characteristics of LAK and NK cells.
   Rabinowich et al., 1993. See above.
  Provides a detailed description of separation of NK cells from PBMC and an example of how purified NK cells are used for studies of NK cell characteristics in vitro.
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